Pyrrolidine derivatives as oxytocin/vasopressin via receptors antagonists

ABSTRACT

The present invention relates to a compound of formula (3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O-meth19243yloxime, and/or an active metabolite thereof having antagonist action at the oxytocin receptor and/or vasopressin V1a receptor, to processes for their preparation, pharmaceutical compositions containing them and their use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of, and claims priority to, U.S.patent application Ser. No. 15/938,729, now U.S. Pat. No. 10,604,482,filed Mar. 28, 2018, which is a continuation of, and claims priority to,U.S. patent application Ser. No. 15/476,325, now U.S. Pat. No.10,047,048, filed Mar. 31, 2017, which is a continuation of, and claimspriority to, U.S. patent application Ser. No. 14/479,664, now U.S. Pat.No. 9,670,155, filed Sep. 8, 2014, which claims priority to EuropeanPatent Application No. 13183723.9, filed Sep. 10, 2013, the contents ofwhich are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof having antagonistaction at the oxytocin receptor and/or vasopressin V1a receptor, toprocesses for their preparation, pharmaceutical compositions containingthem and their use in medicine.

BACKGROUND OF THE INVENTION

Oxytocin (OT) is a cyclic nona-peptide that mediates its physiologicalactions through activation of the oxytocin receptor (OT-R), a cellmembrane receptor belonging to the class of G protein-coupled receptorsthat is similar to arginine vasopressin receptors. One important actionof Oxytocin (OT) is to cause the contraction of the uterus of mammalsduring labor. Repeated, concerted and regular contraction of the uteruswill cause the dilatation of the cervix, the rupture of fetal membranesand lead to expulsion of the fetus. Premature labor is when thesecontractions occur before the normal term of pregnancy. Preterm increaseof uterine activity is the most common expression of preterm labor.

Premature labor leads to undesired premature birth, a serious healthproblem that remains the major cause of perinatal mortality and severemorbidity, especially respiratory distress syndrome, intraventricularhaemorrhage, bronchopulmonary dysplasia and necrotizing enterocolitisthat are far more common in preterm than in term infants. Long-termimpairments such as cerebral palsy, visual impairment and hearing lossare also more common in preterm infants. Nowadays, preterm birth remainsthe leading cause of infant mortality and morbidity in industrializednations, where, despite the significant improvements in obstetricalmedicine, it is causing high costs for neonatal intensive care ofpremature babies. The actual costs are even higher to society whentaking into consideration the healthcare provision of pretermchildbirth-related ailments, such as respiratory distress syndrome,heart conditions, cerebral palsy, epilepsy, and severe learningdisabilities. The management of preterm labor represents a significantproblem in the field of obstetrics.

The OT/OT-R system plays a vital role in initiating labor in mammals, inparticular in humans. The density of OT-R increases markedly in themyometrium before the onset and during labor. Also it is thought thatthe local OT peptide hormone concentration increases markedly beforeparturition in human. The high circulating concentrations ofprogesterone induce uterine quiescence while the uterus acquirescontractile ability. Shortly before term, plasma progesteroneconcentrations fall, OT-R expression in the uterus increases markedly,OT is released and uterine contractile activity increases. At term, thecontractions rise to a crescendo, resulting in delivery as a result oftwo interacting positive feedback loops. The first is a local uterineloop: within the uterus itself, contractile prostaglandins are producedand released in response to OT and uterine contractions. Theseprostaglandins may play a further role in cervical ripening andweakening of fetal membranes. The second loop involves the hypothalamus:in response to uterine contractions and vaginal and cervical distension,magnocellular oxytocin neurons in the hypothalamus increase theiractivity resulting in the release of OT from their axon terminals in theposterior pituitary. The released OT acts upon the uterus both tostimulate the further production of prostaglandins and to contributefurther to the contractions of the uterus.

Therefore, blocking the effect of OT by antagonizing OT-R mightrepresent an attractive modality for the treatment of diseases relatedto the OT-R activity, in particular preterm labor.

Tocolytic, i.e. uterus relaxing agents, have been used in clinicalstudies for the pharmaceutical treatment of preterm labor. Most of theseagents are used off-label. They have shown very limited efficacy, ifany, in prolonging gestation and without clear demonstration ofimprovement of neonate outcome. Current tocolytics are very oftenassociated with unwanted adverse effects on women, foetus or neonate.Such tocolytics include beta-2-adrenergic agonists, prostaglandinsynthesis inhibitors, magnesium sulfate, nitric acid donors and calciumchannel blockers. Beta-2-adrenergic agonists such as ritodrine orterbutaline cause a number of cardiovascular and metabolic side effectsincluding maternal tachycardia, palpitations, hypotension, alteredthyroid function and fetal and neonatal hypoglycaemia, tachycardia.Ritodrine is no longer FDA approved. The calcium channel blockernifedipine is also a medicine that is used to try to stop contractions.Some of the side effects that may occur include facial flushing,headache, nausea, palpitations, and lightheadedness. The totalprostaglandin synthesis inhibitor (NSAID) indomethacin has been used. Itcan also have serious effects on the fetus: constriction of ductusarteriosus, pulmonary hypertension, decrease in renal function witholigohydramnios, intraventricular hemorrhage, hyperbilirubinemia,necrotizing enterocolitis. Maternal side effects include abdominaldiscomfort, nausea, vomiting, depression and dizzy spells for themother. Another NSAID is sulindac that has a side effect profile similarto indomethacin. For magnesium sulfate, meta-analyses have failed tosupport it as a tocolytic agent. Women reported important side effectssuch as flushing, lethargy, headache, muscle weakness, pulmonary edemaand cardiac arrest. A newborn who has been exposed to magnesium sulfatemay show lethargy, hypotonia, respiratory depression, bone problems,osteopenia and fractures. Recently, the FDA is advising healthcareprofessionals against using magnesium sulfate injection for longer than5-7 days to stop preterm labor in women.

Atosiban, a dual vasopressin V1a receptor and OT-R antagonist ismarketed in EU and used to stop contractions and delay preterm deliveryby a few days. Atosiban is a peptide that is not orally bioavailable andmust be administered parenterally. It is rapidly degraded in circulationby enzymes and its use is limited to maximum 48h.

In addition, non-peptide OT-R antagonists were developed such aspyrrolidine derivatives (WO 01/72705, WO 02/102799, WO 2002/074741, WO2004/005249) as mixtures of isomers.

There remain significant unmet needs for efficient and orally selectiveOT-R antagonist for the treatment of diseases related to the OT-Ractivity, in particular preterm labor.

SUMMARY OF THE INVENTION

The present invention provides a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)caxbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof.

The invention also provides a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use as amedicament and pharmaceutical compositions comprising said compound.

Also provided is a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite, for the treatment and/orprevention of disorders associated with the oxytocin receptor activityand/or vasopressin V1a receptor activity.

The invention further provides a process for preparing and isolating acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof in substantially pureform.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B describe dose-response effects of the Z-isomer andE-isomer administered by oral route on inhibition of spontaneous uterinecontractions in anesthetized pregnant rats near term (gestational days19-21). Data as means±S.E. of n=6-8 animals per group. The y-axisrepresents uterine contractions as % of value compared to pre-dose setat 100%. The x-axis represents the time post-dose in minutes.Contractions were continuously recorded and area-under-the-curve (AUC)integrated over 10-min time intervals.

The results presented in FIG. 1A demonstrate that(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (Z form) is capable of rapidly inhibiting spontaneousuterine contractions in anesthetized late-term pregnant rat at variousdoses (10, 30 or 60 mg/kg) compared to control vehicle NP3S (5%N-methylpyrrolidone, 25% polyethyleneglycol 200, 30% polyethylene glycol400,20% propylene glycol, 20% saline). Uterine contractions inhibitionof 15% can be observed 5 to 15 min after administration of thesubstantially pure Z form. Efficient inhibition of 42% is observed170-180 minutes after administration of said compound.

In contrast, no inhibition of uterine contraction has been observed with(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime at various doses (10, 30 or 60 mg/kg, E form) at any timeduring the 170-180 minutes observation (FIG. 1B).

FIG. 2 is a schematic TLC profile showing the results of a dry flashchromatography purification of a crude isomeric mixture of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. As described in Example 1, below, a crude mixture of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime concentrated to dryness was re-dissolved in 2 volumetoluene and loaded onto a pad of SiO2 (S wt) prior to elution using 2Svolume fractions of eluent. Fractions 1-5 were eluted with pure toluene;fractions 6-10 were eluted with toluene/MeOH 1% vol/vol; and fractions10-15 were eluted with toluene/MeOH 2% vol/vol. The Z and E forms areshown by shaded spots. Fractions 8 to 13 were combined and concentratedto dryness. The results show a recovery of 75%. There was no improvementin the E/Z ratio. A minor gain of about 4% area in purity of theisomeric mixture (E+Z) was observed before and after dry-flashchromatography.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, said compound beingin the Z isomeric configuration at the O-methyloxime functional group.

The compound of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime differs from compounds of the present invention at theO-methyloxime functional group being in the E isomeric configuration.

As used herein, the term “active metabolite thereof” refers to a productproduced through metabolism in the body or m vitro of a specifiedcompound, in the present case (3Z,5S)-5-(hydroxymethyl)-1-[(2′-methy1-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O-methyloxime and whichexhibits the same biological activity as(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime.

Active metabolites of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime may be identified using routine techniques known in theart and their activities determined using tests such as those describedherein. Such metabolites may result for example from the oxidation,glucuronation or other conjugation, hydrolysis, reduction and the like,of the administered Z form. Accordingly, the invention includes activemetabolites of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof. Such metabolitemay also be produced in vitro by oxidation, reduction, hydrolysis,glucuronation or other conjugation transformation of the corresponding(3Z,5S)-5-(hydroxymethyl)-1-[(2-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. Examples of actives metabolites of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, include compounds those structures are shown below:

A compound which, upon administration to the recipient, is capable ofbeing converted into a compound of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof as described above,is known as a “prodrug”. A prodrug may, for example, be converted withinthe body, e. g. by hydrolysis in the blood, into its active form thathas medical effects. Pharmaceutical acceptable prodrugs are described inT. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 ofthe A. C. S. Symposium Series (1976); “Design of Prodrugs” ed. H.Bundgaard, Elsevier, 1985; and in Edward B. Roche, ed., BioreversibleCarriers in Drug Design, American Pharmaceutical Association andPergamon Press, 1987, which are incorporated herein by reference.

The compound of the present invention is produced by methods such asthose disclosed for example in WO2004/005249 and WO2005/082848. However,said compound is synthesized and obtained in isomeric mixtures(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime comprising(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime.

Thus, the present invention relates to a compound of formula(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-2-oneO-methyloxime, and/or an active metabolite thereof comprising at least85% to 100% of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and/or metabolite thereof, preferably 85% to 99.9%, morepreferably 90% to 99.9%, and even more preferably 95% to 99.9% of saidcompound.

Alternatively, the present invention relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, provided insubstantially pure form.

As used herein, the term “substantially pure” refers to a compoundprovided in a form which is substantially free of other compounds.Examples of said “other compounds” include(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime,(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one,(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneoxime, (3R,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-3-methoxyamino-pyrrolidine,(3S,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-3-methoxyamino-pyrrolidine,(3Z,5S)-5-(0-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and(3E,5S)-5-(0-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime.

Most preferably, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof is substantially freeof the compound of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime.

Even more preferably, the purity of a substantially pure form compoundof formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, is at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, at least 99%, atleast 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least99.6%, at least 99.7%, at least 99.8%, at least 99.9% or at least 100%and is therefore substantially free of compound of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, i.e. less than 45%, less than 35%, less than 25%, lessthan 15%, less than 10%, less than 5%, less than 3%, more preferablyless than 2%, even more preferably less than 1%.

Even more preferably, the purity of the substantially pure form compoundof formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, is at least in therange of 85% to 100%, preferably 85% to 99.9%, more preferably 90% to99.9%, and even more preferably in the range of 95% to 99.9%.

Depending on the nomenclature used, the compound of the invention“(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime” can also be defined as“(4Z,2S)-2-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl-carbonyl)]pyrrolidine-4-oneO-methyloxime.

Generally, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, is an oxytocinreceptor antagonist.

As used herein, the term “oxytocin receptor antagonist” refers to acompound that functions by inhibiting (partially or completely) orblocking the oxytocin receptor (OT-R), thereby preventing activation ofthe receptor by oxytocin.

The present invention provides a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and/or an active metabolite thereof wherein said compoundis a partial or complete oxytocin receptor antagonist and wherein theinhibitor constant Ki is less than about 1 μM. Preferably, saidinhibitor constant Ki is less than about 0.1 μM, more preferably lessthan about 0.06 μM.

The present invention further provides a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and/or an active metabolite thereof wherein said compoundis an oxytocin receptor antagonist and wherein the half maximalinhibitory concentration IC50 is less than about 1 μM. Preferably, saidIC50 is less than about 0.1 μM, more preferably less than about 0.09 μM.

Generally also, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, is a vasopressin V1areceptor antagonist.

As used herein, the term “vasopressin V1a receptor antagonist” refers toa compound that functions by inhibiting (partially or completely) orblocking the vasopressin V1a receptor (also known as Argininevasopressin receptor 1A), thereby preventing activation of the receptorby vasopressin. Vasopressin V1a receptor is one of the three majorreceptor types for the peptide hormone arginine vasopressin, the othersbeing V1b and V2 receptors

Preferably, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and/or an active metabolite thereof is a vasopressin V1areceptor antagonist, wherein the inhibitor constant Ki is less thanabout 1 μM. Most preferably, said inhibitor constant Ki is less thanabout 0.5 μM, even more preferably less than about 0.15 μM.

The present invention also relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, that is an oxytocinreceptor antagonist and a vasopressin V1a receptor antagonist.

Usually, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, inhibits the uterinecontractions. Advantageously, said compound inhibits uterinecontractions rapidly in a time lapse of 2-30, preferably 5-20 minutesfollowing its administration.

Surprisingly, the Applicants have shown that the inhibitory activity isspecific to the substantially pure Z form of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or metabolite thereof. As shown in the Examples, thesubstantially pure E form of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime shows no efficacy as it does not inhibit the uterinecontractions.

The dosage regimen regarding the compound of the present inventionand/or an active metabolite thereof is selected in accordance with avariety of factors including type, species, age, weight, sex and medicalcondition of the patient; the severity of the condition to be treated;the route of administration; the renal and hepatic function of thepatient; and the particular compound or active metabolite thereofemployed. An ordinarily skilled physician can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Advantageously, a compound of the present invention and/or an activemetabolite thereof may be administered in a single dose, or the totaldosage may be administered in divided doses of two, three or four timesdaily.

Preferably, the present invention provides a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, wherein saidcompound is administered to a subject in a single dose of 50 mg to 900mg, more preferably in a single dose of 100 mg to 600 mg.

Whilst a compound of the invention and/or an active metabolite thereofmay be used as the sole active ingredient in a medicament, it is alsopossible for the compound to be used in combination with at least one ormore further active compounds. Such further active compounds may befurther compounds according to the invention, or other active compoundsselected from the group comprising calcium channel blockers, magnesiumsulfate, selective prostaglandin modulators, beta-2-adrenergic agonists,beta-3-adrenergic receptor agonists, and/or corticosteroids.

Alternatively, the compound of the invention and/or an active metabolitethereof can be administered concomitantly or separately with at leastone compound selected from the group comprising calcium channel blockers(such as nifedipine), magnesium sulfate, prostaglandin receptorsmodulators (such as agonists or antagonists of either EP1 or EP2 or EP3or EP4 or FP receptors), prostaglandin synthesis inhibitors (such asindomethacin, nimesulide, sulindac, rofecoxib, celecoxib),beta-2-adrenergic agonists (such as ritodrine, terbutaline, salbutamol),beta-3-adrenergic receptor agonists, nitric acid donors (such asglyceryl trinitrate) and/or corticosteroids (such as dexamethasone,betamethasone).

As used herein, the term “concomitantly” refers to the administration ofa compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, which is thenimmediately followed by the administration of at least one compoundselected from the group disclosed supra.

As used herein, the term “separately (encompassing sequential orsubsequent administration)” refers to the administration of a compoundof formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, followed by a timeperiod of discontinuance, which is then followed by the administrationof at least one compound disclosed supra.

Generally, the compound of the invention is stable in the plasma. Asused herein the term “stable” refers to the presence of the compound offormula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof in the plasma of thesubject following administration and wherein isomeric interconversion ofsaid compounds is substantially prevented.

Generally, in the present invention the subject in need thereof ispreferably a mammal, most preferably a human, more preferably a woman,and most preferably a human female of child bearing age.

The present invention also relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use as amedicament.

Also envisioned in the present invention is a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use in thetreatment and/or prevention of disorders associated with the oxytocinreceptor activity and/or vasopressin V1a receptor activity.

The disorders associated with the oxytocin receptor activity and/orvasopressin V1a receptor activity are selected from the non-limitinggroup comprising preterm labor, premature birth, dysmenorrhea, prematureejaculation, sexual dysfunction, endometriosis, embryo implantationfailure due to uterine contractions, infertility, benign prostatichyperplasia, neuro-psychiatric disorders, autism, social behaviordisorders, psycho-social stress, and/or cardiovascular disorders.

The term “preterm labor” referring also to premature labor, shall meanexpulsion from the uterus of a viable infant before the normal end ofgestation, or more particularly, onset of labor with effacement anddilation of the cervix before the 37th week of gestation. It may or maynot be associated with vaginal bleeding or rupture of the membranes.

The term “dysmenorrhea” refers to a condition characterized by cyclicpain associated with menses during ovulatory cycles. The pain is thoughtto result from uterine contractions and ischemia.

The term “sexual dysfunction” refers to any disturbance or variation inthe four phases—excitement phase, plateau phase, orgasmic phase andresolution phase characterizing the human sexual response.

The term “neuro-psychiatric disorders” as used herein refers to mentaldisorders attributable to diseases of the nervous system, e.g.depression, obsessive-compulsive disorder and others.

The term “social behavior disorders” as used herein refers to emotionaldisturbance, inappropriate types of behavior or feelings, pervasive moodof unhappiness or depression and a range of perceived difficulties tobuild or maintain satisfactory interpersonal relationships.

The term “psycho-social stress” as used herein refers to a conditionresulting from a perceived threat to the social status, social esteem,self-worth, respect or acceptance within a group, and that lead todevelopment of a stress response in the body and physical symptoms.

Assisted reproduce on technologies are methods applied in humans for thetreatment of infertility and in animals for producing pregnancies.Infertility, which affects about 10% of human pairs worldwide, may betreated by in vitro fertilization and embryo transfer (IVF-ET) or inless complicated cases, by artificial insemination. Generally, a successof an embryo transfer is dependent on uterine receptivity, an entitythat is defined as an ability of uterus to provide optimal conditionsmandating proper implantation and embryo development. Basic componentsof uterine receptivity are uterine contractile activity and thecondition of endometrium.

Uterine contractions occurring during the embryo transfer may expelembryos from the uterus towards vagina or oviducts, which may be a causeof unsuccessful treatment, or in latter case a cause of extrauterinepregnancy, a serious, potentially life-threatening complication.

Generally, the present invention relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use in assistedreproduction technology.

For example, the present invention relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use in thetreatment of infertility by in vitro fertilization-embryo transfer(IVF-ET) method.

The present invention also relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use in reducingembryo implantation failure due to uterine contractions.

Also envisioned in the present invention is a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use in reducingcontractions occurring during the embryo transfer.

Furthermore, the present invention relates to a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, for use in thetreatment and/or prevention of a disease relating to oxytocin-inducedvascular contractility, vasopressin-induced vascular contractility,oxytocin-induced muscular contractility, vasopressin-induced muscularcontractility.

The present invention further relates to a pharmaceutical compositioncomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, and apharmaceutically acceptable carrier, diluent or excipient.

A “pharmaceutically acceptable carrier, diluent or excipient” usedherein is a medium generally accepted in the art for the delivery ofbiologically active agents to patients. A person skilled in the art isaware of a whole variety of such carriers, diluents or excipientssuitable to formulate a pharmaceutical composition (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Publishing Company,1990, pp. 1289-1329). The carriers), diluent(s) or excipient(s) must becompatible with the other ingredients of the formulation, capable ofpharmaceutical formulation, and not deleterious to the recipientthereof.

The compound of the invention and/or an active metabolite thereof,together with a conventionally employed earner, diluent or excipient maybe formulated as pharmaceutical compositions and unit dosages thereof,and in such form may be employed as solids, such as tablets or filledcapsules, or liquids such as solutions, suspensions, emulsions, elixirs,or capsules filled with the same, all for oral use, or in the form ofsterile injectable solutions for parenteral (including subcutaneous)use. Such pharmaceutical compositions and unit dosage forms thereof maycomprise ingredients in conventional proportions, with or withoutadditional active compounds or principles, and such unit dosage formsmay contain any suitable effective amount of the active ingredient, i.e.the compound of the invention, commensurate with the intended dailydosage range to be employed.

The pharmaceutical compositions of the invention can be administered bya variety of routes including oral, rectal, vaginal, transdermal,subcutaneous, intravenous, intramuscular, and intranasal. Depending onthe intended route of delivery, the compounds are preferably formulatedas either injectable or oral compositions. The compositions for oraladministration can take the form of bulk liquid solutions orsuspensions, or bulk powders. More commonly, however, the compositionsare presented in unit dosage forms to facilitate accurate dosing. Theterm “unit dosage forms” refers to physically discrete units suitable asunitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical excipient. Typical unit dosage forms include prefilled,premeasured ampoules or syringes of the liquid compositions or pills,tablets, capsules or the like in the case of solid compositions. In suchcompositions, the compound of the invention is usually a minor component(from about 0.1 to about 50% by weight or preferably from about 1 toabout 40% by weight) with the remainder being various vehicles orcarriers and processing aids helpful for forming the desired dosingform.

Preferably, the pharmaceutical composition comprising a compound offormula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, and apharmaceutically acceptable carrier, diluent or excipient isadministered by oral, vaginal or intravenous route.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatine; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas pepper-mint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the aft.

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can also befound in Gennaro, A. R. et al, Remington's Pharmaceutical Sciences. 18thed. Eastern: The Mack Publishing Company, 1995.

The present invention also relates to a process for preparing andisolating the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, in substantiallypure form comprising the steps of:

a) Loading a crude isomeric mixture comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, on a gelchromatography column;

b) Purifying with 1% alcohol in organic solvent; and

c) Purifying with 2% alcohol in organic solvent.

As used herein, the term “crude isomeric mixture” refers to a mixture ofcompounds resulting from the synthesis of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, as described hereinand comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and a compound of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime.

Preferably, the invention relates to a process for preparing andisolating the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, in substantiallypure form comprising the steps of:

a) Loading a crude isomeric mixture comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, and/or an active metabolite thereof, on a silica gelchromatography column;

b) Purifying with 1% methanol in toluene

c) Purifying with 2% methanol in toluene

Preferably, the silica gel chromatography column is chosen from Biotage®Flash ISO flash chromatography system, Biotage KP-SIL, BiotageKP-C18-HS, Biotage KP-C18-WP, Biotage KP-C-WP, Biotage FLASH-WAC 400(Biotage AB, 751 03 Uppsala, Sweden). Other gel chromatography columnsinclude columns loaded with Mitsubishi Diaion™ HP20 or HP20SS SDVBresins (Mitsubishi Chemical Corporation, Tokyo 100-8251, Japan).

EXAMPLES Example 1: Purification of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime 1.1 Synthesis of(3Z/P5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime

The present invention relates to the synthesis and purification of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime obtained as a crude isomeric mixture comprising(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime.

Synthetic pathways of compounds of the invention are for example thosedescribed in WO2004005249 and WO2005082848.

For example, compound of the invention(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime can also be prepared following stages 1 to 7 as describedbelow:

Stage 1: Preparation of 4-(2-methylphenyl)benzoic acid

A solution of potassium carbonate (0.908 Kg, 6.57 mol, 2.06 wt) in water(2.20 L, 5.0 vol) was charged to a slurry of 4-bromobenzoic acid (0.441Kg, 2.19 mol, 1.0 wt) in water (4.41 L, 15.0 vol) at 15 to 25° C. Theresulting slurry was stirred at 15 to 25° C. and degassed three timesusing a vacuum-nitrogen purge cycle.Tetrakis(triphenylphosphine)palladium(0) (0.022 Kg, 0.019 mol, 0.05 wt)was charged and the vacuum-nitrogen purge cycle repeated. A solution ofo-tolylboronic acid (0.313 Kg, 2.30 mol, 0.707 wt) in methanol (3.53 L,8.0 vol) was degassed three times, using a vacuum-nitrogen purge cycle,and then charged to the 4-bromobenzoic acid slurry at 15 to 25° C. Thereaction mixture was heated to and maintained at reflux (71 to 78° C.)until reaction completion (The reaction is considered complete at 95%conversion), as determined by ‘H NMR analysis (d6-DMSO), typically 1.5to 2.5 hours. The reaction mixture was concentrated to 15 vol undervacuum at 40 to 45° C. Toluene (4.41 L, 10.0 vol) and tetrahydrofuran(4.41 L, 10.0 vol) were added to the residue, the resulting mixturestirred vigorously and acidified to pH 1 with hydrochloric acid (6M,2.00 L, 4.5 vol). The contents were stirred vigorously for 30 to 60minutes and the layers separated. Toluene (2.20 L, 5.0 vol) andtetrahydrofuran (2.20 L, 5.0 vol) were added to the aqueous phase andthe mixture stirred for 5 to 10 minutes. The layers were separated, thecombined organic phases filtered and concentrated to 10.0 vol undervacuum at 35 to 40° C. Toluene (4.41 L, 10.0 vol) was added to theresidue and the resultant concentrated under vacuum at 35 to 40° C. Thetetrahydrofuran content of the resulting slurry was determined by ‘H NMRanalysis (d6-DMSO) (Pass level: ≤1.0% w/w tetrahydrofuran with respectto toluene). The slurry was coded to and aged at 0 to 5° C. for 30 to 60minutes, the solid collected by filtration and the filter-cake washedwith toluene (2.20 L, 5.0 vol). The solid was dried in a vacuum oven at35 to 40° C. to give 4-(2-methylphenyl)benzoic acid [0.438 Kg, 94.1% th,99.3% w/w, 1H NMR (d6-DMSO) concordant with structure] as a pale yellowsolid.

Stage 2: Preparation of 4-(2-methylphenyl)benzoic acid chloride

Thionyl chloride (0.300 L, 4.11 md, 0.685 vol) was added to a slurry of4-(2-methylphenyl)benzoic acid (0.435 Kg, 2.05 mol, 1.0 wt) in toluene(4.35 L, 10.0 vol) at 10 to 25° C. and the mixture heated to andmaintained at 75 to 80° C.3 until complete by 1H NMR analysis(d6-benzene), typically 4 to 5 hours. Reaction completion wasaccompanied by the formation of a hazy solution. The resultant wasconcentrated to 5.0 vol by removal of toluene under reduced pressure at35 to 45° C. Toluene (2.18 L, 5.0 vol) was added to the concentrate andthe mixture concentrated to 4.0 vol by removal of toluene under reducedpressure at 35 to 45° C. The resultant was filtered through glassmicrofiber paper and the filter-cake washed with toluene (0.44 L, 1.0vol). The toluene solution of 4-(2-methylphenyl)benzoic acid chloride[0.439 Kg, 92.8% th, 100.9% w/w, 1H NMR (d6-benzene) concordant withstructure] was used directly in Stage 3.

Stage 3: Preparation of(4R)-4-hydroxy-1-[(2′-methyl-1,1′-biphenyl-4yl)-carbonyl]-L-proline

A solution of potassium carbonate (0.526 Kg, 3.31 mol, 1.2 wt) in water(0.57 L, 1.3 vol) was charged to a solution of 4-hydroxy-L-proline(0.274 Kg, 2.09 mol, 0.625 wt) in tetrahydrofuran (2.20 L, 5.0 vol) andwater (0.44 L, 1.0 vol) at 15 to 25° C. followed by a line rinse ofwater (0.44 L, 1.0 vol). The mixture was cooled to 0 to 5° C. with rapidstirring and a solution of 4-(2-methylphenyl)benzoic acid chloride(0.438 Kg, 1.90 mol, 1.0 wt) in toluene (2.19 L, 5.0 vol) charged atthat temperature followed by a line rinse of toluene (0.44 L, 1.0 vol).The reaction mixture was warmed to 15 to 25° C. over 1 to 2 hours andstirred at this temperature until judged complete by TLC analysis. Water(2.20 L, 5.0 vol) was charged to the reaction mixture at 15 to 25° C.and the layers separated. The aqueous phase was acidified to pH 5 to 6with aq. hydrochloric acid (6M, 0.66 L, 1.5 vol) and then to pH1 withaq. hydrochloric acid (2M, 0.88 L, 2.0 vol) at 15 to 25° C. The mixturewas cooled to and aged at 0 to 5° C. for 30 to 60 minutes, theprecipitated solid collected by filtration, the filter-cake washed withwater (2×1.75 L, 2×4.0 vol) and toluene (0.88 L, 2.0 vol) and pulled dryon the filter for 12 to 24 hours. The collected solid was dried undervacuum at 40 to 45° C. until the water content by KF was ≤0.2% w/w toafford(4R)-4-hydroxy-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline[0.599 Kg, 97.0% th, 136.8% w/w, ¹H NMR (d₆-DMSO) concordant withstructure] as an off-white solid.

Stage 4: Preparation of1-(2′-methyl-1,1′-biphenyl-4-yl)carbonyl-4-oxo-L-proline

Triethylamine (1.80 L, 13.56 mol, 3.0 vol) was charged to a solution of(4R)-4-hydroxy-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline(0.598 Kg, 1.84 mol, 1.0 wt) in dimethyl sulfoxide (4.42 L, 7.4 vol) at15 to 20° C. Pyridine-sulphur trioxide complex (0.879 Kg, 5.52 mol, 1.47wt) was charged portion-wise at 15 and 25° C. and the reaction mixturestirred at that temperature until reaction completion, as determined byTLC analysis (typically 1 to 3 hours).7 The reaction was quenched withaq. hydrochloric acid (3M, 4.80 L, 8.0 vol) at 0 to 30° C.,tetrahydrofuran (3.00 L, 5.0 vol) and heptanes (0.60 L, 1.0 vol)charged, the layers separated and the aqueous phase extracted withtetrahydrofuran (2×3.00 L, 2×5.0 vol). The combined organic phases werewashed with aq. hydrochloric acid (1 M, 2×1.20 L, 2×2.0 vol) andsaturated sodium chloride solution (2×1 20 L, 2×2.0 vol), the aqueouswashes combined and back-extracted with tetrahydrofuran (2×0.60 L, 2×1.0vol). The combined organics were dried over magnesium sulphate (1.794Kg, 3.0 wt), filtered, the filtercake washed with tetrahydrofuran (0.60L, 1.0 vol) and the filtrates concentrated under vacuum at 40 to 45° C.to give a pale brown foam. Ethyl acetate (6.00 L, 10.0 vol) was chargedto the foam, the contents stirred for 5 to 10 minutes to reachdissolution and the solvent removed under vacuum at 40 to 45° C. Thiswas repeated using ethyl acetate (6.00 L, 5.0 vol) until tetrahydrofuranwas not detected by ¹H NMR analysis (d₆-DMSO). The residue was slurriedin ethyl acetate (4.80 L, 8.0 vol), activated carbon (0.084 Kg, 0.14 wt)added followed by a line rinse of ethyl acetate (3.00 L, 5.0 vol), theresultant heated to and maintained at 70 to 80° C. for 20 to 30 minutes,cooled to 40 to 55° C. and filtered through glass microfiber paper. Thefilter-cake was washed with ethyl acetate (1.50 L, 2.5 vol) and thecombined filtrates and wash concentrated to 2.5 to 3.5 vol under vacuumat 40 to 45° C. Crystallisation commenced during the concentration. Theconcentrate was transferred to a suitable vessel with a line rinse ofethyl acetate (0.30 L, 0.5 vol) and heated to 70 to 80° C. Additionalethyl acetate (0.30 L, 0.5 vol) was added as necessary to achievedissolution. Heptanes (1.80 L, 3.0 vol) was added at 70 to 80° C. andthe contents allowed to cool to between 15 and 25° C. over 1 to 2 hours.The slurry was further cooled to and aged at 0 to 5° C. for 2 to 3hours, filtered and the filter cake washed with ethyl acetate:heptanes(1:1, 0.60 L, 1.0 vol) at 0 to 5° C. followed by heptanes (3.0 L, 2.5vol). The collected solid was dried under vacuum at 40 to 45° C. to give1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-4-oxo-L-proline [0.444 Kg,74.7% th, 74.2% w/w, ¹H NMR (d₆-DMSO) concordant with structure] as anoff-white solid.

Stage 5: Preparation of(4Z/E)-4-methoxyimino-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline

Triethylamine (0.40 L, 2.85 mol, 0.92 vol) was added to a solution of1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-4-oxo-L-proline (0.434 Kg,1.34 mol, 1.0 wt) in dichloromethane (4.40 L, 10.0 vol) at 10 to 25° C.followed by a line rinse of dichloromethane (0.43 L, 1.0 vol).Methoxylamine hydrochloride (0.130 Kg, 1.56 mol, 0.30 wt) was addedportionwise at 10 to 25° C. followed by a line rinse of dichloromethane(0.43 L, 1.0 vol) and the reaction mixture stirred at 10 to 25° C. untilreaction completion, as determined by TLC analysis (typically 3 to 5hours, TLC eluent: dichloromethane:methanol:acetic acid (90:10:1); uvvisualization). The solvent was removed under vacuum at 35 to 40° C.,the resultant dissolved in ethyl acetate (4.40 L, 10.0 vol) and washedwith aq. hydrochloric acid (1 M, 2×2.20 L, 2×5.0 vol). The acidic washeswere back extracted with ethyl acetate (2.20 L, 5.0 vol), the combinedorganic phases washed with sat. aq. sodium chloride solution (3.10 L,7.0 vol), dried over magnesium sulfate (0.300 Kg, 0.69 wt), filtered andthe filtercake washed with ethyl acetate (2.20 L, 5.0 vol). The filtrateand washes were combined and concentrated under vacuum at 35 to 40° C.to afford4-methoxyimino-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline[0.476 Kg, 100.6% th, 109.6% w/w, ¹H NMR (CDCl₃) concordant withstructure) as an off-white solid.

Stage 6: Preparation of (4Z/E,2S)-methyl-1-[(2′-methyl-1,1′-biphenyl]-4-yl)-carbonyl-4-methoxyiminopyrrolidine-2-carboxylate

Potassium carbonate (0.476 Kg, 3.44 mol, 1.0 wt) was added to a solutionof 4-methoxyimino-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline(0.475 Kg, 1.35 mol, 1.0 wt) in acetone (4.75 L, 10.0 vol) and themixture cooled to 0 to 10° C. Dimethyl sulfate (0.128 L, 1.35 mol, 0.27vol) was added at 0 to 15° C. and the mixture stirred at 15 to 25° C.until reaction completion, as determined by TLC analysis, typically 3 to16 hours. The solvent was removed under vacuum at 40 to 45° C. and theresultant partitioned between ethyl acetate (3.80 L, 8.0 vol) and water(3.80 L, 8.0 vol). The layers were separated, the organic phase washedwith sat. aq. sodium chloride solution (2.85 L, 6.0 vol), dried oversodium sulfate (0.953 Kg, 2.0 wt) and filtered. The filter-cake waswashed with ethyl acetate (0.48 L, 1.0 vol) and the combined filtrateand wash concentrated under vacuum at 40 to 45° C. Excess ethyl acetatewas removed by azeotropic distillation with tetrahydrofuran (2×0.9SL,2×2.0 vol) under vacuum at 40 to 45° C. to give (4Z/E,2S)-methyl-1-[(2′-methyl-1,1′-biphenyl-4-yl)-carbonyl]-4-methoxyiminopyrrolidine-2-carboxylate [0.492 Kg, 99.6% th, 103.6% w/w, ¹H NMR(CDCl₃) concordant with structure] as a viscous brown oil.

Stage 7: Preparation of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime

Lithium borohydride (0.049 Kg, 2.26 mol, 0.1 wt) was added portionwiseunder nitrogen to a stirred solution of (4Z/E,2S)-methyl-1-[(2′-methyl-1,1′-biphenyl-4-yl)-carbonyl]-4-methoxyiminopyrrolidine-2-carboxylate (0.492 Kg, 1.34 mol, 1.0 wt) intetrahydrofuran (2.31 L, 4.7 vol) and methanol (2.31 L, 4.7 vol) at 0 to30° C. The mixture was stirred at 15 to 25° C. to reaction completion,as determined by TLC analysis (Eluent: ethyl acetate; Visualisation:ninhydrin), typically 2 to 6 hours. The reaction mixture was quenchedwith water (0.40 L, 0.8 val) at 15 to 25° C. and stirred at 15 to 25° C.for 16 to 20 hours. The resultant was concentrated under vacuum at 40 to45° C. and the residue partitioned between water (2.46 L, 5.0 vol) andethyl acetate (4.92 L, 10.0 vol). The layers were separated, the organicphase washed sequentially with aq. hydrochloric acid (1M, 2.46 L, 5.0vol), sat. aq. sodium hydrogen carbonate solution (2.46 L, 5.0 vol) andsat. aq. sodium chloride solution (2.46 L, 5.0 vol). The organic phasewas dried over magnesium sulfate (0.985 Kg, 2.0 wt), filtered and thefilter-cake washed with ethyl acetate (0.50 L, 1.0 vol). The combinedfiltrate and wash were concentrated under vacuum to give a crudeisomeric mixture comprising(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime [0.395 Kg, 86.9% th, 80.3% w/w, 1H NMR (CDCl₃) concordantwith structure; 82.0% area by HPLC, 71.4:28.6 Z/E ratio] as a viscousbrown oil. The oil was dissolved in toluene (0.40 L, 1.0 vol, withrespect to weight of product) and stored until required.

1.2 Dry flash chromatography of crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime

A dry flash chromatography purification of the crude isomeric mixtureobtained following the protocol described above was attempted usingdifferent elution conditions. A crude mixture of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime concentrated to dryness was re-dissolved in 2 volumetoluene and loaded onto a pad of SiO2 (S wt) prior to elution using 25volume fractions of eluent.

Fractions 1-5: eluted with pure toluene

Fractions 6-10: eluted with Toluene/MeOH 1% vol/vol

Fractions 10 to 15: eluted with Toluene/MeOH 2% vol/vol

The Z and E forms are shown by shaded spots. Fractions 8 to 13 werecombined and concentrated to dryness. The results show a recovery of75%. There was no improvement in the E/Z ratio. A minor gain of about 4%area in purity of the isomeric mixture (E+Z) was observed before andafter dry-flash chromatography (Table I).

TABLE I Comparative impurity profile before and after dry-flashchromatography % area Impurity at E + Z- Impurity at RRT 1.12 RRT 0.7isomers RRT 1.08 (Ar—Ar—CH2OH) Before dry 4.6 91.3 <0.5 4.1 flash Afterdry- 2.5 95.6 <0.5 0.7 flash RRT: Relative retention time

The dry-flash chromatography of the crude isomeric mixture does notallow the purification of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. The E/Z ratio pre and post dry-flash remain in the rangeof 30/70 to 40/60.

Furthermore, such an approach should be considered on the basis of thescale at which the operation has to be carried out. On a 20 L scale,this operation would not be a time saving approach.

1.3 Assessment Toward Crystallization of the Pure Z from the CrudeIsomeric Mixture

The first part of the assessment toward crystallisation of the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime from the crude mixture(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, has been looking at solubility and possiblecrystallisation conditions of the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. The results of the solubility/crystallisation testscarried out on 15 mg scale are reported in Table II below

TABLE II Qualitative solubility data for(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O-methyloxime SolventDissolves in: Comment heptanes — insoluble in 20 vol toluene 2 vol coldDIPE 40 vol hot THF 4 vol cold tBuOH 6 vol hot MIBK 4 vol hot IPA 4 volhot

The initial solubility screen showed that pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime isomer is soluble in a range of solvents. On the basis ofthe above results, crystallisation by addition of anti-solvent wasexamined and the results reported in Table III. The anti-solvent wasadded to a warm solution ca 40-50° C. and allowed to cool to roomtemperature.

In particular, the water (anti-solvent) was added to a warm (40-50° C.)solution of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime in IPA until cloudiness was reached and the mixture wasallowed to cool to room temperature.

TABLE III Crystallisation via addition of anti-solvent SolventAntisolvent Comment toluene 20 vol heptanes 39 vol oils out THF 10 volheptanes 40 vol oils out tBuOH 10 vol water 20 vol oils out MIBK 10 volheptanes 40 vol oils out IPA 20 vol water 160 vol very fine solid, oilsout on standing IPA 8 vol water 18 vol very fine solid, oils out onstanding DMSO 10 vol water 12 vol gel NMP 10 vol water 28 vol oils outMeOH 10 vol water 10 vol oils out DMSO 20 vol water 16 vol oils outacetone 10 vol water 10 vol oils out DCM 10 vol heptanes 50 vol oils out

The IPA/water crystallisation conditions were applied to a crudeisomeric mixture. The toluene solution was first concentrated to drynessprior to dissolution in IPA (8 vol) and addition of water (18 vol).Unfortunately, this resulted in material de-mixing as dl.

In another experiment, the antisolvent was added to a solution of crude(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (90.4% area purity, contained 0.5% w/w toluene and 3.7%w/w THF) at room temperature until cloudiness was reached and themixture was left to stand at room temperature (Table IV).

TABLE IV Crystallisation by addition of water at 18-22° C. SolventAntisolvent Comment MeOH 5 vol water 3 vol oils out DMSO 5 vol water 3vol oils out

At this point of the investigation, no suitable conditions ofcrystallisation of the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime or allowing isolation of solid containing(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime have been identified.

Further crystallisation attempts were carried out using crude isomericmixture of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. In all cases, the volume of solvents was smaller thanwhat used previously and based only on a single solvent. The crudematerial (E/Z ratio 33:67 and purity (E+Z) 79.52% area) used for thiscrystallisation was concentrated to a foam (Table V).

TABLE V crystallisation from single solvent at lower volume MaterialSolvent Ageing in freezer Ageing in fridge ‘Pure Z’ Ethyl Crystallisesre- Stays in solution, with Acetate dissolves as warms and withoutseeding 1.8 vol after 2 days. Crude Does not crystallize n/a with orwithout seeding. ‘Pure Z’ Diethylether On addition of ether n/a 2.3 volat 18-22° C. starts to dissolve then crashes out again. Recovery 70%Used for seeding Crude Oils Crystallises recovery 41 Re-dissolves as %E/Z ratio 40/60 purity warms 85.4% area. (mother liquors E/Z ratio 20/80purity 62.1% area). Seeds not used. ‘Pure Z’ TBME Oils Stays insolution, with 2.3 vol Re-dissolves as and without seeding warms after 2days. Crude Oils Stays in solution, with Re-dissolves as and withoutseeding warms after 2 days.

Crystallisation using ethyl acetate followed by aging in a freezerovernight gave crystallisation using the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime material, but quickly re-dissolved as the sample warmed.No crystals were observed using crude material in ethyl acetate evenwhen seeds were added.

Crystallisation using diethylether followed by aging in a fridge gavecrystallisation using the crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime material. The solid was collected in 41% recovery.Unfortunately, the collected solid had a slighter poorer E/Z ratio thanthe input material and a slightly higher chemical purity.

TBME as solvent for both pure Z and crude gave oiling after aging infreezer, and stayed in solution after aging in the fridge with andwithout seeds.

Suitable crystallization conditions of the crude isomeric mixtureallowing improvement of the Z/E ratio and of the purity of the isomericmixture (5+Z) have not been found.

1.4 Substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime 1.4.1 Small Scale Purification

The isolation procedure in substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime was performed by chromatography using a Biotage system(Biotage AB, SE-751 03 Uppsala, Sweden) of the crude isomeric mixtureisolated after reduction of the oxime ester (Stage 7 of Example 1).

Five distinct batches (No. 020, 180, 062, 068, 076) of the crudeisomeric mixture were purified by Biotage chromatography. Furthermore,different conditions were used regarding batches No. 068 and 076.Purification was performed with a 5% w/w spike of oxime methyl esteradded (No. 068), and with an overloaded Biotage column (No. 076).

Each chromatography was run using Biotage 40M cartridges (40 g silica)which had been pre-flushed with toluene. Toluene:MeOH (99:1 v/v) wasthen eluted and collected in 100 ml fractions (total volume 4 L),followed by a flush of toluene:MeOH (96:4 v/v).

Fractions were analysed by TLC (eluent: ethylacetate) to determine whichfractions could be discarded and which fractions contained Z isomer.These Z fractions were then analyzed by HPLC. The pass criteria for afraction was >96% Z isomer and <1.2% E isomer.

Surprisingly, the purification through Biotage chromatography of variousbatches was very efficient as the substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is purified at 99.4% (Batches No. 020, No. 062, No. 068)and at 99.2% (Batches No. 180, No. 076). In particular, the Biotagechromatography in presence of oxime ester removes 5% w/w oxime esterwithout detriment to recovery or quality (Batch No. 068) and a 25%overcharge of the Biotage column does not cause a decrease in yield orquality (batch No. 076).

TABLE VI efficiency of the Biotage chromatography Batch yield of No.Input % E/Z Output % E/Z Z isomer 020 3.0 g Pure Z-fractions: 33% 85.7%area purity 1.0 g % E/Z: 30.5/69.5 98.8% area purity % E/Z: 0.6/99.4 1802.0 g Pure Z-fractions 45% 92.0% area purity 0.9 g % E/Z: 32.8/67.299.6% area purity % E/Z: 0.8/99.2 062 3.0 g Pure Z-fractions 43% 83.5%area purity 1.3 g % E/Z: 32.7/67.3 99.8% area purity % E/Z: 0.6/99.4Mixture: 11% 1.2 g 91.0% area purity % E/Z: 69.6/30.4 068 3.0 g spikedwith ~5% Pure Z fractions: 40% ester 1.2 g ~78% area purity 99.8% areapurity % E/Z: 32.7/67.3 % E/Z: 0.6/99.4 Mixture: 14% 0.6 g 98.8% areapurity % E/Z: 27.9/72.1 Pure E fractions: N/A 1.1 g 70.7% area purity %E/Z: 98.7/1.3 (19.3% ester) 076 3.8 g Pure Z fractions 37% 83.5% areapurity 1.4 g % E/Z: 32.7/67.3 99.8% area purity % E/Z: 0.8/99.2 Mixture:17% 1.8 g 95.0% area purity % E/Z: 63.6/36.4

1.4.2 Large Scale Purification

Various batches of crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (0.392 kg, 1.16 mol, 1.0 wt) were charged to a Biotage 150L SIM unit as an approximate 50% w/w solution in toluene and purifiedusing 1% methanol in toluene (150 L) followed by 2% methanol in toluene(50 L), fraction size 5.0 L. The collected fractions were analysed byTLC¹⁵ and HPLC analyses, as appropriate. The fractions that were deemedto contain clean(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (criteria: Z-isomer ≥96.00% area. E-isomer ≤1.20% area)were combined and concentrated under vacuum at 40 to 45° C. Absoluteethanol (2× 2 L) was added to the residue and the solution concentratedunder vacuum at 40 to 45° C. until the foamy solid could be manipulated.The desired product,(3Z,5S)-1-[(biphenyl-4-yl-carbonyl)-5-hydroxy-methyl]pyrrolidine-3-one-O-methyloxime(0.089 Kg, 22.7% w/w, ¹H NMR (CDCl₃) concordant with structure, 99.3%area by HPLC, 98, 4:0.9 Z/E ratio was obtained as an off-white to lightbrown solid.

TABLE VII Summary of purification of different batches of (3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime in substantially pure form. Batch Input Output Yield% Z form % E form No. (kg) (kg) (% w/w) (% area) (% area) 12 0.392 0.08922.8 98.65 0.85 116 0.392 0.114 29 98.34 0.89 120 0.441 0.081 18.4 97.901.81 122 0.380 0.094 24.3 98.52 1.14 124 0.387 0.096 25.3 98.89 0.73 1260.390 0.132 33.8 98.40 0.95 128 0.526 0.010 2 98.20 0.83 130 0.453 0.08619 98.46 1.23 132 0.440 0.082 19.3 98.86 0.85 134 0.39 0.144 36.9 98.730.96 138 0.273 0.098 35.9 98.92 0.66 140 0.463 0.059 13.1 98.52 1.13 1420.462 0.084 18.4 99.37 0.48 144 0.442 0.126 29 99.1 0.68 146 0.409 0.13533.5 99.21 0.46 148 0.460 0.107 23.8 99.13 0.65 150 0.409 0.071 18 98.920.66 152 0.392 0.054 14.3 98.82 0.76 156 0.445 0.039 8.8 98.64 0.87 1580.392 0.06 15.3 98.73 0.63 162 0.435 0.150 34.5 98.94 0.79 164 0.4340.192 44.2 99.21 0.58 166 0.415 0.074 17.8 98.79 0.73 174 0.518 0.10820.8 99.11 0.64 176 0.342 0.072 21 98.88 0.77 178 0.415 0.074 17.8 99.070.71 180 0.353 0.174 49.3 99.03 0.82 182 0.270 0.178 65.9 99.10 0.53

Appropriate batches of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (2.713 kg, 1.0 wt) isolated from the Biotagechromatography were combined and dissolved in absolute ethanol (5.16 L,2.0 vol) at 15 to 25° C., clarified by filtration through glassmicrofiber paper and an absolute ethanol wash (0.50 L, 0.2 vol) appliedto the filter. The combined filtrates were concentrated portion wiseunder vacuum at 40 to 45° C. The resultant was transferred to dryingtrays and dried under vacuum at 30° C. for 24 hours. The oventemperature was then increased incrementally from 30 to 40° C. over 80hours. The level of residual solvent was determined by ¹H NMR analysis(CDCl₃) and when found to be <1.0% w/w the solid was passed through a500 μm aperture sieve. The solid was returned to the oven and dried at40 to 42° C. until the solvent level was ≤0.40% w/w to afford(3Z,5S)-1-[(biphenyl-4-yl-carbonyl)-5-hydroxy-methyl]-pyrrolidine-3-one-O-methyloxime(2.633 Kg, 97.1% w/w, 1H NMR (CDCI3) concordant with structure, 98.65%area by HPLC.

The combination procedure is summarized below:

Input: 2.713 kg

Output: 2.633 kg

Yield: 97.1% w/w

Example 2:(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime binding to OT-R and vasopressin V1a receptor

Binding to the OT-R and Vasopressin V1a receptor

Competition binding to the human oxytocin receptor was measured in vitrousing a scintillation proximity assay.

This assay allows determining the affinity of the test compounds forOT-R. Membranes from HEK293EBNA (cells expressing OT-R) were suspendedin buffer containing 50 mM Tris-HCl, pH 7.4.5 mM MgCl2 and 0.1% BSA(w/v). The membranes (2-4 μg) were mixed with 0.1 mg SPA bead coatedwith wheat-germ agglutinin (WGA-PVT-Polyethylene Imine beads fromAmersham) and 0.2 nM of the 125 radiolabelled [I]-OVTA (OVTA beingOrnithin Vasoactive, an analogue of OT for competitive bindingexperiments). Non-specific binding was determined in the presence of 1μM Oxytocin. The total assay volume was 100 μl. The plates (Corning® NBSplate) were incubated at room temperature for 30 min and counted on aMibrobeta® plate scintillation counter. Competitive binding wasperformed in presence of compounds of the present invention at thefollowing concentrations: 30 μM, 10 μM, 1 μM, 300 nM, 100 nM, 10 nM, 1nM, 100 μM, 10 μM. The competitive binding data were analysed using theiterative, nonlinear, curve-fitting program, “Prism” (GraphPad Software,Ine).

The ability of the compounds of the present invention to inhibit thebinding of I-OVTA to the OT-receptor was assessed using the abovedescribed in vitro biological assay. The binding affinity of testcompounds from the above examples is expressed by the inhibitionconstant (Ki; nM). From these values, it can be derived that said testcompounds according to the present invention do show a significantbinding to the oxytocin receptor.

The inhibition constant Ki of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime to the oxytocin receptor is Ki (nM)=52 nM and to thevasopressin V1a receptor is Ki (nM)=120 nM. Thus(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is selective for the oxytocin receptor.

Example 3:(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is a OT-R antagonist

The inhibition of oxytocin-induced Ca2+ mobilization in OT-R transfectedHEK293EBNA cells was measured by FLIPR (fiuorimetric imaging platereader). This assay allows the measurement of the inhibition of OT/OT-Rmediated calcium mobilization by the compound(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime.

FLIPR® is a fluorimetric imaging device using a laser (Argon-ion laser)for simultaneous illumination and reading (cooled CCD camera) of eachwell of a 96-well-plate, thus enabling rapid measurements on a largenumber of samples.

Preparing the plates: FLIPR-plates were pre-coated with PLL(Poly-L-Lysine) 10 μg/ml+0.1%) gelatine to attach HEK293EBNA cells(Human Embryonic Kidney cells expressing OT-R) and incubated for 30 minup to 2 days at 37° C. The cells were plated out into 96-well-plates(60000 cells/well).

Labelling with fluo-4: 50 μg of Fluo-4 (Ca2+ sensitive fluorescent dye)were dissolved in 20 μl pluronic acid (20% in DMSO). The dissolvedfluo-4 was then diluted in 10 ml DMEM (Dubecco's Minimal EssentialMedium)-F12 culture medium. The plates were washed one time withDMEM-F12 medium. 100 μl of the Fluo-4 containing-DMEM-F12 medium wereadded to the HEK-cells which were incubated for 1.5-2h in thisfluorescent medium. Fluo-4 is taken up by the cytoplasm of the cells.

Buffer: 145 mM NaCl, 5 mM KCl, ImM MgCl2, 10 mM Hepes, 10 mM Glucose,EGTA (Ethylene-bis oxyethylene nitrilo tetraacetic acid). The pH wasadjusted to 7.4.

Performance of the assay: A minimum of 80 μl/well of compound(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (5×) in the above buffer (1×) were prepared(96-well-plates). The compound(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime was added to the 96-well-plates at differentconcentrations (30 μM, 10 μM, 1 μM, 300 nM, 100 nM, 10 nM, 1 nM, 100 μM,10 μM). Oxytocin (OT) was added at a concentration of 40 nM.

The relative fluorescence of Fluo-4 (λex=488 nm, λem=590 nm) is thenmeasured by the FLIPR in presence or absence of compounds(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. The fluorescence of the marker being sensitive to theamount of Ca2+, the Ca2+ movements can be detected. Then, it can bedetermined the ability of compound(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime to antagonize the oxytocin-induced intracellular Ca2+mobilization mediated by the oxytocin receptor.

The compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime inhibits the activity of oxytocin on OT-R with an IC50=81nM.

Example 4: Inhibition of Spontaneous Uterine Contractions inAnesthetized Late-Term Pregnant Rat 4.1 Experimental Protocol

Late-term pregnant (certified at days 19-21 of pregnancy) Sprague DawleyCD (SD) BR female rats (Charles River, Calco, Italy), weighing 350-400 gwere anesthetized with urethane (1.05 g/kg, i.p.) and placed on ahomoeothermic operating table. A midline incision at the hypogastriumlevel was made, one pregnant uterine horn exposed and its tubal end(near the ovary) was closed by a ligature with surgical silk.

Corresponding to the fetus close to the ovary, the uterine-horn wall wasincised taking care not to injure the adjacent placenta and PE240 tubingwith a latex balloon (9 mm length when empty, capacity 0.1 mL; Radnoti,Monrovia, Calif., USA) on the top was inserted into the lumen andsecured to the uterine wall with surgical silk. After filling theinternal cavity of the latex balloon with 0.1 mL of sterilephysiological saline solution, the catheter was connected to anamplifying/recording system (MacLab, ADInstruments Pty Ltd, Castle Hill,Australia) via a P23ID Gould-Statham pressure transducer. One jugularvein was isolated and cannulated with a PE60 polyethylene cannula forthe i.v. administration. After the surgical preparation, a 30-minstabilization period was observed and then the effects of increasingdoses of compounds of the present invention (given as 10-min i.v.infusion, bolus i.v. or p.o.) were evaluated by measuring the resultinguterine contractions.

For the i.v. administration (infusion or bolus) the uterine contractileactivity was quantified by calculating the AUC during the 10-mininjection period. The percent variation of the AUC values relative tothe spontaneous uterine response observed after each compoundadministration was calculated in comparison to the value recorded beforethe first dose-administration (basal value).

The effect of test compounds of the present invention was evaluated bycomparing pre- and post-treatment luminal uterine pressure values.

For the oral administration the same computation procedure was appliedat different time points after treatment. Statistical differencesbetween treatment groups at each time-point were determined by usingone-way ANOVA followed by Tukey test.

4.2 Results

FIGS. 1A and B describe dose-response effects of Z-isomer and E-isomeradministered by oral route on inhibition of spontaneous uterinecontractions in anesthetized pregnant rats near term (gestational days19-21). Data as means±S.E. of n=6-8 animals per group. The y-axisrepresents uterine contractions as % of value compared to pre-dose setat 100%. The x-axis represents the time post-dose in minutes.Contractions were continuously recorded and area-under-the-curve (AUC)integrated over 10-min time intervals.

The results presented on FIG. 1A demonstrate that(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (Z form) is capable to rapidly inhibit spontaneous uterinecontractions in anesthetized late-term pregnant rat at various doses(10, 30 or 60 mg/kg) compared to control vehicle NP3S (5%N-methylpyrrolidone, 25% polyethyleneglycol 200, 30% polyethylene glycol400,20% propylene glycol, 20% saline). Uterine contractions inhibitionof 15% can be observed 5 to 15 min after administration of thesubstantially pure Z form. Efficient inhibition of 42% is observed170-180 minutes after administration of said compound.

In contrast, no inhibition of uterine contraction has been observed with(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime at various doses (10, 30 or 60 mg/kg, E form) at any timeduring the 170-180 minutes observation (FIG. 1B).

Surprisingly, the present invention shows that the substantially pure Zform having formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime inhibits uterine contraction whereas the substantiallypure E form has no efficacy. Thus, the present invention advantageouslyrelates to biologically active compounds of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and/or metabolite thereof in substantially pure form thatcan be administered at lower dosage compared to said compounds providedin isomeric mixture.

Example 5: In vivo stability of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime

The isomeric interconversion of[¹⁴C]-(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime was studied after single oral and intravenous doses(nominal 30 mg/kg, 25 μCi/rat) to eight healthy female rats (n=4 foreach dose route).

The animals used in this study were Spague-Dawley. Crl: CD®BR albinotats. All animals were supplied by Charles River UK Ltd (Margate, Kent,UK). Animals were in the weight range 200-260 g and were approximately 2months old. RMS were given a unique identity number and were identifiedby unique tail markings plus cage location.

The dose groups were as follows: 4 female were given an oral dose and 4female were given an intravenous dose.

[¹⁴C]-(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrolidin-3-oneO-methyloxime oral and intravenous dosing formulations were preparedseparately at each dose phase at a target dose level of 30 mg/kg, and ata radioactive concentration of approximately 25 μCi/rat. Doseformulations were prepared in an appropriate matrix; intravenous doseswere prepared in NP3S, whilst oral doses were prepared in Labrasol:water(1:1 v/v).

Chromatographic analysis using HPLC indicated that radioactivecomponents exhibiting co-chromatography with the E-isomer of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime were not present in the oral or intravenous doseformulations either pre- or post-dose administration. There wastherefore no detectable interconversion of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime to(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime daring dose preparation or administration. There was noevidence that [¹⁴C]-E-isomer of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime was present in plasma collected up to 6 hours after anoral or intravenous administration of[¹⁴C]-(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime.

Therefore, using the methods described[¹⁴C]-(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime undergoes no detectable isomeric interconversion in vivoafter oral or intravenous dose administration.

The invention claimed is:
 1. A pharmaceutical composition comprising (i)a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and (ii) one or more pharmaceutically acceptable carriers,diluents, or excipients, wherein the purity of the compound relative toone or more other stereoisomers of the compound is at least 85%.
 2. Thepharmaceutical composition of claim 1, wherein the purity of thecompound relative to one or more other stereoisomers of the compound isat least 90%.
 3. The pharmaceutical composition of claim 2, wherein thepurity of the compound relative to one or more other stereoisomers ofthe compound is at least 95%.
 4. The pharmaceutical composition of claim1, wherein the purity of the compound relative to one or more otherstereoisomers of the compound is from about 90% to about 99.9%.
 5. Thepharmaceutical composition of claim 4, wherein the purity of thecompound relative to one or more other stereoisomers of the compound isfrom about 95% to about 99.9%.
 6. A pharmaceutical compositioncomprising (i) a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and (ii) one or more pharmaceutically acceptable carriers,diluents, or excipients, wherein the purity of the compound relative toits stereoisomer,(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, is at least 85%.
 7. The pharmaceutical composition ofclaim 6, wherein the purity of the compound relative to(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is at least 90%.
 8. The pharmaceutical composition ofclaim 7, wherein the purity of the compound relative to(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is at least 95%.
 9. The pharmaceutical composition ofclaim 6, wherein the purity of the compound relative to(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is from about 90% to about 99.9%.
 10. The pharmaceuticalcomposition of claim 9, wherein the purity of the compound relative to(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is from about 95% to about 99.9%.